Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme

Ritwika S Basu,Brittany A Warner,Vadim Molodtsov,Danil Pupov,Daria Esyunina,Carlos Fernández-Tornero,Andrey Kulbachinskiy,Katsuhiko S Murakami

doi:10.1074/jbc.m114.584037

Abstract

The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.

Highlights

Cellular RNA polymerases start transcription by de novo RNA priming
We report the structures of bacterial transcription initiation complexes with initiating ribonucleoside triphosphates (iNTPs) or short RNA product bound in the active site of RNA polymerase (RNAP)
The binding of the first iNTP is a special feature of all cellular RNAPs that are capable of primer-independent de novo transcription initiation

Summary

Background

Cellular RNA polymerases start transcription by de novo RNA priming. Results: Structures and biochemical studies of initially transcribing complexes elucidate the de novo transcription initiation and early stage of RNA transcription. All cellular and most bacteriophage RNAPs initiate transcription de novo by loading two iNTPs opposite the first and second template DNA bases at the transcription start site to synthesize the first phosphodiester bond of RNA. The roles of the observed RNAP-iNTP contacts in transcription initiation were not tested experimentally; in addition, the structure contained a suboptimal template strand sequence around the transcription start site (see below), suggesting that it might miss some important contacts with the iNTPs. Following the de novo incorporation step, RNAP goes through several cycles of NTP addition before entering transcription elongation, during which a highly stable and proces-. We prepared a homogeneous initially transcribing complex with a 6-mer RNA using in crystallo transcription approach and solved its crystal structure, which provides insights into the binding of short RNA-DNA hybrid in the active site and in the process of ␴ release triggered by the nascent RNA, as the initially transcribing complex begins transition into the elongation phase of transcription

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION